Water and Quartz

Langmuir 2002, 18, 2807-2811

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Investigations of Lysozyme Adsorption at the Air/Water and Quartz/Water Interfaces by Vibrational Sum Frequency Spectroscopy Gibum Kim, Marc Gurau, Joonyeong Kim, and Paul S. Cremer* Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77843-3012 Received August 20, 2001. In Final Form: December 28, 2001 We used vibrational sum frequency spectroscopy (VSFS) to investigate lysozyme adsorption at the quartz/water and air/water interfaces. These investigations found that water structure depended intimately on bulk pH at both the hydrophobic, air, and hydrophilic, quartz, interfaces. Changes in the interfacial potential caused by protonation/deprotonation of titratable groups on the protein (as well as on quartz in the case of the solid/liquid studies) played a major role in this. Furthermore, intensity in the CH stretch range from lysozyme was observed under all conditions investigated at the air/water interface. Because only moieties that are well-aligned at the interface can be observed in VSFS, this was taken to be an indication that at least some lysozyme residues must be organized at the hydrophobic air surface. By contrast, CH stretches were not observed from protein adsorbed at the hydrophilic quartz/water interface unless the pH was raised above 8.0, causing the interface to take on a net negative charge. Even under very basic conditions, however, the CH stretch modes were much weaker in this case than those observed at the hydrophobic air/water interface.

Introduction Adsorption of proteins at interfaces has been a topic of increasing interest over the past several decades1-3 because of its importance in fields ranging from medical implantation4 and drug delivery5,6 to biological sensor fabrication.7,8 A variety of techniques have been employed to investigate adsorption behavior both at the solid/ liquid9-15 and vapor/liquid13,16-19 interfaces. One protein * To whom correspondence should be addressed. Tel.: 979-8621200. Fax: 979-845-7561. E-mail: [email protected]. (1) Proteins at Interfaces: Physicochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series 343; American Chemical Society: Washington, DC, 1987. (2) Proteins at Interfaces II: Fundamentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series 602; American Chemical Society: Washington, DC, 1995. (3) Tertykh, V. A.; Yanishpolskii, V. V. In Adsorption on Silica Surfaces; Papirer, E., Ed.; Surfactant Science Series; Marcel Dekker: New York, 2000; Vol. 90, p 523. (4) Kenausis, G. L.; Voros, J.; Elbert, D. L.; Huang, N.; Hofer, R.; Ruiz-Taylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 3298. (5) Bae, Y. H.; Okano, T.; Kim, S. W. J. Polym. Sci., Polym. Phys. 1990, 28, 923. (6) Malmsten, M. Colloids Surf., A 1999, 159, 77. (7) Jones, V. W.; Kenseth, J. R.; Porter, M. D.; Mosher, C. L.; Henderson, E. Anal. Chem. 1998, 70, 1233. (8) Seigel, R. R.; Harder, P.; Dahint, R.; Grunze, M. Anal. Chem. 1997, 69, 3321. (9) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87. (10) Wahlgren, M.; Arnebrant, T.; Lundstrom, I. J. Colloid Interface Sci. 1995, 175, 506. (11) Robenson, J. L.; Tilton, R. D. Langmuir 1996, 12, 6104. (12) Norde, W.; Rouwendal, E. J. Colloid Interface Sci. 1990, 139, 169. (13) Ball, A.; Jones, R. A. L. Langmuir 1995, 11, 3542. (14) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. J. Phys. Chem. B. 1998, 102, 8100. (15) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438. (16) Razumovsky, L.; Damodaran, S. Langmuir 1999, 15, 1392. (17) Graham, D. E.; Philips, M. C. J. Colloid Interface Sci. 1979, 70, 427. (18) Postel, C.; Abillon, O. Langmuir 1998, 14, 5649. (19) Lu, J. R.; Su, T. J.; Howlin, B. J. J. Phys. Chem. B 1999, 103, 5903.

that has received particular attention is hen egg white lysozyme because the structural characteristics of this macromolecule are well-understood at the molecular level.20,21 Lysozyme is composed of 129 amino acids, including 18 cationic and 12 anionic residues.22 Lysozyme is considered to be a hard protein because four internal disulfide bonds help maintain its tertiary structure.23 The attention afforded lysozyme has already allowed many of its adsorption properties to be elucidated. For example, it is known that maximum protein adsorption at the air/water interface occurs near pH 11.0, the isoelectric point of this protein.19 Furthermore, several studies23-25 showed changes in surface tension as a function of time after the adsorption of lysozyme. These data were interpreted as the unfolding or partial denaturation of the protein caused by contact with the hydrophobic air surface. Other studies showed that a decrease in enzymatic activity was concomitant with this process.26 It has also been revealed by neutron reflectivity and ellipsometry that the global conformation of the macromolecule can change from a “side-on” to a “headon” orientation with respect to the molecule’s long axis as the interfacial density of the protein is increased.19,27 The neutron reflectivity data also found no evidence for gross denaturation of the protein based upon layer thickness measurements.19 (20) Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.; Sarma, V. R. Nature 1965, 206, 757. (21) Shirahama, H.; Lyklema, J.; Norde, W. J. Colloid Interface Sci. 1990, 139, 177. (22) Clenfield, R. E.; Liu, A. K. J. Biol. Chem. 1965, 240, 2000. (23) Tripp, B.; Magda, J. J.; Andrade, J. D. J. Colloid Interface Sci. 1995, 173, 16. (24) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403. (25) Xu, S.; Damaodaran, S. J. Colloid Interface Sci. 1993, 159, 124. (26) Hunter, J. R.; Kilpatrick, P. K.; Carbonell, R. G. J. Colloid Interface Sci. 1990, 137. (27) Horsley, D.; Herron, J.; Hlady, V.; Andrade, J. D. In Proteins at Interfaces: Physicochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; ACS Symposium Series 343; American Chemical Society, Washington, DC, 1987; Chapter 19.

10.1021/la0113365 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/07/2002

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Langmuir, Vol. 18, No. 7, 2002

Figure 1. Schematic side-cut view of the flow cell in the VSFS experimental setup: (a) IR-grade fused quartz window; (b) Teflon flow cell body; (c) protein solution reservoir; (d) pH meter; and (e) drain from flow cell. The flow cell was replaced with a Langmuir trough for air/water interface studies.

In addition to air/water interface investigations, lysozyme adsorption studies have been carried out at the liquid/solid interface on fused quartz substrates. It was shown that adsorption also increased substantially in this case as the pH of the bulk solution was tuned through the isoelectric point.15,28 Total internal reflection fluorescence microscopy11 and attenuated total internal reflection FTIR studies13 showed that conformational changes occurred on quartz substrates even below the isoelectric point of the protein. Furthermore, the amount of helical content of the protein decreased.29 Finally, reorientation from “side-on” to “head-on” adsorption was observed in neutron reflectivity studies depending on the specific surface density.15 In this paper we have employed vibrational sum frequency spectroscopy (VSFS), a surface-specific nonlinear optical technique, to investigate lysozyme adsorption at both the quartz/water and air/water interfaces. VSFS is a particularly valuable technique for such studies because it is capable of monitoring the reorganization of interfacial water molecules upon the surface recruitment of a variety of adsorbates such as proteins,30 polyelectrolytes,31 and lipid bilayers32 even in the presence of an overwhelming contribution of bulk water. Our investigations revealed that water structure was particularly sensitive to pH changes at both the hydrophobic, air, and hydrophilic, quartz, interfaces. Furthermore, intensity in the CH stretch range from lysozyme itself was observed under all air/water interface conditions investigated. This was a clear indication that hydrophobic lysozyme residues become aligned at this hydrophobic interface. More surprisingly, weak CH stretch intensity could also be observed from protein adsorption at the quartz/water interface, but only under high pH conditions where the overall surface charge was negative. Experimental Section Instrumentation. VSFS experiments were performed with a passive-active mode-locked Nd:YAG laser (PY61c, Continuum, Santa Clara, CA) equipped with a negative feedback loop in the oscillator cavity to provide enhanced shot-to-shot stability. The 1064-nm beam had a pulse width of 21 ps and operated at a repetition rate of 20 Hz. It was used to pump an optical parametric generator/oscillator (OPG/OPA) stage (Laser Vision, Bellevue, WA) that generated the 532 nm and tunable infrared input beams (2800 to 4000 cm-1) used in these experiments. Figure 1 shows the setup of the VSFS beams and sample stage. The IR and the visible beams were concentrically overlapped at the sample (28) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267. (29) Norde, W.; Giacomelli, C. E. Macromol. Symp. 1999, 145, 125. (30) Kim, J.; Cremer, P. S. ChemPhysChem 2001, 8/9, 543. (31) Kim, J.; Cremer, P. S. J. Am. Chem. Soc. 2000, 122, 12371. (32) Kim, J.; Kim, G.; Cremer, P. S. Langmuir 2001, 17, 7255.

Kim et al. interface with incident angles of 51° and 42°, respectively, with respect to the surface normal. All sum frequency spectra presented in this article have been taken with the SSP polarization combination, referring to the sum frequency, visible, and infrared beams, respectively. Each data set was normalized to spectra taken from a piece of Y-cut crystalline quartz. Both the experimental and theoretical details of vibrational sum frequency spectroscopy have been described elsewhere.33-35 Briefly, a sum frequency response is obtained, in the dipole approximation, only in media that lack inversion symmetry. In systems where the bulk is isotropic, the overwhelming majority of the signal is obtained at interfacial boundaries where inversion symmetry is necessarily broken. Vibrational spectra are obtained in VSFS experiments through resonance enhancements that occur as the infrared beam is tuned over vibrational modes of the surface species. As shown in eq 1, the sum frequency signal, ISFS, is proportional to the square of the second-order nonlinear susceptibility, χ(2). χ(2) can be broken into two parts, a frequency(2) dependent resonant term, χ(2) R , and nonresonant term, χNR, (2) 2 ISFS ∝ |χ(2)|2IvisIIR ) |χ(2) R + χNR| IvisIIR

(1)

where Ivis and IIR denote the visible and IR beam intensities, respectively. The resonant term can be expressed as follows,

χR(2)n )

∑ω n

An IR

- ωn + iΓn

(2)

where An, ωn, ωIR, and Γn are the oscillator strength, resonant frequency, frequency of the IR beam, and damping constant of the nth vibrational resonant mode, respectively. An is the product of the infrared and Raman transition dipole moments as well as a phase factor, their orientational vector average, and their number density at the interface. This is essentially the heart of the sum frequency selection rules giving rise to interfacial sensitivity. Namely, the vast majority of the signal is derived from an ordering of dipoles, which occurs readily at interfaces. Materials. Purification of lysozyme was accomplished by size exclusion chromatography with a Sephadex G-50 column. After sample preparation, gel electrophoresis showed the presence of only 1 band at about 11 400 Da corresponding to the molecular weight of lysozyme. Polyglycine (molecular weight 4600) and polyalanine (molecular weight 1850) were used as received. The protein, polypeptides, and column were all purchased from Sigma. The concentrations of lysozyme solutions were determined by absorption measurements at 280 nm in a UV/visible spectrometer. The water used in preparing all buffers and cleaning solutions for these experiments was obtained from a NANOpure Ultrapure Water System (Barnstead, Dubuque, IA) and had a minimum resistivity of 18 MΩ‚cm. The D2O (ISOTEC Inc., Miamisburg, OH) used was 99.9% isotopically pure. Buffers at the desired pH values were made by dissolving appropriate amounts of sodium phosphate into solution. The final pH was adjusted to within 0.1 pH units of the desired value by adding HCl or NaOH. Sufficient NaCl was added to all buffers to raise the total ionic strength to 0.030 M. Because the concentration of lysozyme added to the buffers was always 1 mg/mL, it added